Background
Diabetes mellitus (DM) is the most common endocrine disorder, and affects more than 100 million people worldwide (6% of the population) and in the next 10 years it may affect five times more people than it does now (World Health Organization and American Diabetes Association). The World Health Organization has pointed out that the prevention of diabetes and its complications is not only a major challenge for the future, but essential if health for all is to be an attainable target, and strongly emphasize the optimal, rational use of traditional and natural indigenous medicines (World Health Organization 1985, 1994).
There is an imbalance between radical generating and radical scavenging mechanisms i.e increased free radical production or abridged activity of antioxidant defenses or both results in oxidative stress. Oxidative stress is currently suggested as the mechanism underlying diabetes and diabetic complications [
1]. Some of the researches have shown that administration of streptozotocin (STZ) in the animals engender diabetes mellitus and produce an assortment of reactive oxygen species (ROS) as a result of glucose auto-oxidation and protein glycosylation such as superoxide, hydrogen peroxide, and hydroxyl radicals, that are either formed by STZ itself over the short term or result from induced hyperglycemia[
2‐
4]. Additionally, there is a formation of advanced glycation end-products (AGE) by non-enzymatic glycation reactions such as Amadori, Schiff base, and Maillard, persuade the formation of free radical at accelerated rates during the course of diabetes, and are associated with the pathogenesis of chronic diseases such as arthritis, atherosclerosis, and liver cirrhosis [
5,
6]. Consequently, in recent times, antioxidant therapy has been thought to be effectual for the prevention and treatment of various diseases including diabetes, because oxidative stress plays a key role in the pathogenesis of human diseases [
7,
8].
Albizzia Lebbeck Benth. is a deciduous tree with compound leaves and flat oblong fruits. It is distributed throughout India from the plains upto 900 m in the Himalayas. The bark and flowers of
Albizia Lebbeck Benth. were used to treat arthritis according to the Siddha system of Medicine [
9]. Several studies reported the traditional use of A. Lebbeck Benth. such as the tribal people in Himachal Pradesh and Kashmir use the plant to treat inflammation [
10‐
12], while the tribals of Tamilnadu utilizes the plant in the treatment of bone fractures [
13]. Diaorrhea, edema, poisoning, asthma and bronchitis were also being cured by the use of this plant [
14,
15]. Earlier studies also reported the beneficial effects of A. Lebbeck Benth such as the plant reduces the level of histamine and raised the plasma cortisol in antigen challenged guinea pigs [
16] and proves advantageous activity in bronchial asthma patients. [
17]. An anti-inflammatory effect of methanolic extract of
Albizia Lebbeck bark was also reported. [
18,
19]. The antioxidant potential of leaves of
A. Lebbeck Benth. was reported by Resmi et al (2006) [
20]. Furthermore, a recent research work has reported the hypoglycemic action of
Albizzia Lebbeck Benth. bark on diabetic rats. The study confirms the improved glycemic control of
Albizzia Lebbeck Benth. bark [
21]. A research work indicating the antidiabetic potential of
Albizzia Lebbeck bark in alloxan induced diabetic mice was reported [
22]. One report portraying the antidiabetic activity of another important species of
Albizzia i.e. Albizzia odoratissima Benth. in alloxan induced diabetic rats. The study depicted the hypoglycemic potential of Albizzia odoratissima Benth. in diabetic rats [
23]
. The antioxidant action of
Albizzia Lebbeck leaves on alloxan induced diabetic rats was evidenced by another study, confirming the antioxidant activity of
Albizzia Lebbeck Benth. on alloxan induced diabetic rats [
24]. Some other researches that shows the leaves of plant has the antioxidant potential [
25] that can target the free radicals accountable for the destruction of β-cells of pancreas. Consequently, aqueous extract of flowers of
Albizzia Lebbeck showed enhanced glycemic control in alloxan induced diabetic rats [
26].
Despite a long traditional utilization and some reports on the hypoglycemic and antioxidant action of A. Lebbeck Benth. in diabetes, no systematic phytochemical and pharmacological research exertion has been carried out on exhaustive research exertion on mode of action, antihyperlipidemic, pancreas, renal, liver and cardiac histopathological alterations, of the methanol/dichloromethane stem bark extract of this impending plant. Therefore, we have taken this research exertion in order to scrutinize plausible mode of action of anti-diabetic potential and the anti-oxidant action and of the A. Lebbeck Benth. bark.
Methods
Chemicals
Streptozotocin (STZ) was purchased from Sigma Aldrich, St. Louis, USA. The kits for the assay of blood glucose (GLU), total cholesterol(TC), triglyceride(TG), high density Lipoprotein cholesterol(HDL–C), low density lipoprotein cholesterol(LDL–C), hepatic glycogen, hepatic hexokinase, glucose-6-phsophatase, fructose-1-6-bisphosphatase, glucose-6-phosphate, lipid peroxidation (LPO), superoxide dismutase(SOD), catalase (CAT), glutathione Peroxidase (GSH-Px), reduced glutathione (GSH) diagnostic kits were purchased from Span Diagnostics, Surat, India. Glycated serum protein (GSP), blood urea nitrogen (BUN), and creatinine (CRE) diagnostic kits were procured from Accurex, India. Glibenclamide was a generous gift from Ranbaxy Pharmaceutical Company, Gurgaon, India. All other commercial reagents used were of analytical grade.
Animals
Male albino rats aged between 8-10 weeks (250-300 g) were purchased from Indian Institute of Toxicological Research (IITR), Lucknow, UP, India. Animals were kept in controlled condition in animal house at an ambient temperature of 25-30°C and relative humidity of 55-60% and 12/12 h light/dark cycle and were provided pellet diet along with water ad libitum. The experimental protocol has been duly approved by institutional animal ethical committee of Adina Institute of Pharmaceutical Sciences (IAEC Reg. no. 1546/PO/a/11/CPCSEA) and was performed according to the animal ethical guidelines of CPCSEA, government of India.
Plant material
Fresh stem bark pieces of of A. Lebbeck Benth. were collected from herbal garden of faculty of health sciences, SHIATS, Allahabad, between September 2013-October 2013. The stem barks were identified and authenticated by taxonomist, Botany department in FHS, SHIATS, Allahabad, as stem barks of A. Lebbeck. A voucher specimen of the plant (Ref no. FHS/PHCD/ALB/2013-2014/188) has been deposited in the University’s Botany department herbarium.
The A. Lebbeck Benth. stem barks were chopped into small pieces, powdered, air-dried, sieved (#40) and stored in air tight container at room temperature. Two kilogram of powdered plant material was soaked in 4 L of methanol/dichloromethane in a glass jar for two days at room temperature. The mixture was then subjected to the maceration. The solvent was then filtered with Whatman No. 1. The filtration was repeated 4-5 times until the extract depicts no faint discoloration. The yield of the methanol/dichloromethane extract was found to be 10.82% w/w (132 g). Five grams of this extract were dissolved in 1 mL of dimethyl sulfoxide (DMSO) and then the solution adjusted to 100 mL with distilled water. The extract was further subjected to concentration using a rotary evaporator (Buchi, India) under reduced pressure. The extract was freeze dried for further phytochemical screening.
Preliminary phytochemicals studies
The extract was subjected to various phytochemicals tests to determine the active constituents present in the crude methanol/dichloromethane leaves extracts of stem bark of A. Lebbeck Benth.
Acute toxicity study
Acute oral toxicity study was performed according to the 423 guidelines (Acute toxicity class method) lay down by OECD (Organisation of Economic Cooperation and Development). Healthy male albino rats were randomly divided into eight groups with 6 animals in each group. The animals were kept fasting overnight with supplementation of water, thereafter with methanolic/dichloromethane extract of A. Lebbeck Benth. stem bark with increasing doses (100, 200, 300, 400, 500, 600, 700 & 800 mg/kg body weight) with the aid of intragastric tube in order to determine the safe doses by up and down staircase method [
27]. The animals were scrutinized continuously for 1 h, then repeatedly for 4 h and later at the end of 24 h for general behavior, autonomic and neurological profiles. Thereafter, one group was administered high dose of A. Lebbeck Benth. extract orally once daily for 20 days and observed for any lethality and death.
Induction of diabetes
Wistar rats were injected intraperitoneally with STZ dissolved in 0.1 M citrate buffer (pH = 6.5) at 60 mg/kg. Animals of control group were received equal volume of vehicle. After 48 hours of STZ injection, blood glucose of the induced rats was estimated. The rats depicting FBG ≥ 230 mg/dL considered to be diabetic.
Experimental design
A total of 30 male albino wistar rats were utilized and the animals were randomly divided into 7 groups of 5 animals in each group:
Group I- Normal rats (untreated with dimethylsulfoxide, [DMSO, 3 ml/kg])
Group-II- Diabetic control (administered with Streptozotocin (STZ)
Group-III- Diabetic control + A. Lebbeck Benth. stem bark (ALEx) (100 mg/kg body weight)
Group –IV- Diabetic control + A. Lebbeck Benth. stem bark (ALEx) (200 mg/kg body weight)
Group-V- Diabetic control + A. Lebbeck Benth. stem bark (ALEx) (300 mg/kg body weight)
Group-VI- Diabetic control + A. Lebbeck Benth. stem bark (ALEx) (400 mg/kg body weight)
Group-VII- Diabetic control + Glibenclamide (1 mg/kg body weight)
The extract was administered to the respective groups through oral route using intragastric tube for 45 days.
Biochemical evaluation
Rats of the different groups were fasted overnight and the blood was withdrawn by retro orbital puncture under light and under anesthesia. Blood was withdrawn from the rats on the 1st, 22nd and 45th day after the induction of diabetes to assess the blood glucose and plasma insulin level by glucose oxidase method [
28] and modified method of Herbert et al. (1965) [
29] respectively. The alteration in the body weight was observed throughout the therapy in the experimental animals.
At the termination of treatment i.e 45 days, animals were deprived of food for overnight. Activities of hepatic hexokinase, glucose-6-phsophatase, fructose-1-6-bisphosphatase, glucose-6-phosphate were assayed according to the method of Branstrup et al (1957) King (1965), Gancedo and Gancedo (1971) and Robert Langdon (1966), respectively [
30‐
33]. The lipid parameters viz. total cholesterol, HDL cholesterol and triglycerides were evaluated according the method of Zlatkis et al. (1953), Burnstein et al. (1970) and Foster and Dunn (1973), respectively [
34‐
36]. Level of serum LDL cholesterol and VLDL cholesterol were estimated according to the Friedewald formula [
37]. Hepatic glycogen level was assessed by the method given by Kemp and Van Hejnigen (1954) [
38]. The levels of lipid peroxidation (LPO) in the tissues were evaluated by the method of Okhawa et al. (1979) [
39]. Level of superoxide dismutase (SOD) was assayed by the method of Kakkar et al. (1984) [
40]. The level of catalase (CAT) enzyme was evaluated by the method of Sinha et al. (1972) [
41]. Glutathione Peroxidase (GSH-Px) was assayed by the method given by Rotruck et al. (1973) [
42]. Level of reduced glutathione (GSH) was assessed by the method of Ellman (1959) [
43].
Levels of blood urea nitrogen (BUN), glycated serum protein (GSP) and creatinine (CRE) in serum were evaluated according to the manufacturer’s instructions provided in diagnostic kits.
Production of liver and kidney homogenate
For the estimation of the antioxidant level, the rats of the respective groups were kept overnight fasted. All the rats were decapitated and an abdominal incision was performed, in order to harvest liver and pancreas. The whole organs were thoroughly cleaned with chilled normal saline on ice. A 10% (w/v) homogenate of the liver and pancreas (0.03 M sodium phosphate buffer, pH-7.4) was prepared with the help of Ultra-Turrax homogenizer maintaining the speed at 9500 rpm.
Observation of general condition of rats
The overall general condition of rats such as psychological activity, food intake, water intake, urine output, general locomotor activity, and skin infection were observed every day. The parameters such as body weight and food intake were determined every week.
Histological assessment of liver, kidney, pancreas and heart sample by heamatoxylin eosin (H/E) staining
At the end of the treatment with the drug, all the rats of different groups were sacrificed using mild anesthesia. After collection of the blood samples, the liver, kidney, pancreas and heart tissues were fixed in neutral formalin solution for 48 hours, dehydrated by passing through graded series of alcohol embedded in paraffin blocks. 4 μm thick sections were prepared using a semi-automated rotator microtome.
Statistical analysis
Statistical analysis was performed using GRAPH PAD Prism software package, Version 5.0. All the data were expressed as mean ± standard error mean (SEM). The comparisons within groups were evaluated utilizing independent student T-test and one way analysis of variance (ANOVA). The value of p < 0.05 or p < 0.01 were considered to be statistically significant.
Discussion
The present research exertion was designed to evaluate the prospective effects of
Albizzia Lebbeck Benth. stem bark extract (ALEx) on glycemic control, antioxidant status and its histopathological changes on the liver, pancreas, kidney and heart. STZ diabetic model is one of the important and most widely accepted and utilized method to induced diabetes comparable to human diabetes. At present, a growing apprehension has attracted attention of many researchers to and has brought back traditional and complementary medicine due to their pharmacological and economic advantages [
44‐
46]. Our previous research work also depicts the protective effect of one traditionally used polyherbal formulation against the diabetes induced liver and pancreatic damage [
47].
Streptozotocin enters the pancreatic β-cells through one of the important glucose transporter known as GLUT 2 and damages the β-cells by DNA alkylation. Furthermore, the damage is also done by the production of superoxide radicals inside the β-cells which are produced with the help of xanthine oxidase. In addition to the following mechanism another important mechanism by which the β-cells are partially destroyed are the formation of nitric oxide free radicals. Therefore, free radicals play an important role in the development of diabetes mellitus by causing the partial destruction of β-cells [
48]. In view of that, we hypothesized that free radicals scavenging properties of a compound can ameliorate the diabetic conditions.
Flavonoids are naturally occurring phenolic compounds that are found in plants. They are widely distributed in most of the frequently consumed beverages and food products of plant origin such as fruits, vegetables, tea, wine and cocoa [
49]. In recent years much of the attraction was on the antioxidant activity of flavonoids, due to their ability to reduce the free radicals formation and to scavenge free radicals. There are strong experimental evidences that show that patients with diabetes mellitus are susceptible to increase in blood level of oxidants [
50,
51].
An enhancement of blood glucose level was observed in the oral glucose tolerance test (OGTT) was considerably greater in the STZ induced diabetic rats as compared to the non-diabetic rats. The level of plasma insulin was increased in the non-diabetic rats as compared to the diabetic rats in which there is a decrease in the plasma insulin level. Administration of ALEx at different dose noticeably enhanced the impaired glucose tolerance in the STZ induced diabetic rats with improvement in the plasma insulin level. Based on the above results, the hypoglycemic action of the
Albizzia Lebbeck Benth.. stem bark extract may be due to the insulin like action i.e performing its action at the peripheral level to improve the cellular uptake of glucose or enhance the glycogenesis. Many of the plant and their extracts have shown to exert hypoglycemic action through stimulation of insulin release [
52,
53]. The hypoglycemic action of the ALEx is comparable to the conventional sulfonylurea i.e. Glibenclamide that is reported to enhance the insulin release from the beta cells of pancreas though their activation. Therefore, it is presupposed that ALEx could be accountable for potentiation of the pancreatic secretion of insulin from regenerated β-cells by inhibiting ATP sensitive K + channels like Glibenclamide for stimulation of insulin from the pancreatic beta cells.
Diabetic state is characterized by a severe loss in body weight because of loss or degradation of structural proteins [
54]. Due to insulin deficiency there is a marked reduction in the protein content in the muscular tissue due to proteolysis [
55]. The reversal in loss of weight in the ALEx treated diabetic rats group exhibited that restoration of the weight loss may be due the reversal of proteolysis, gluconeogenesis and glycogenolysis [
56].
In experimental diabetes, there is a marked alteration of the enzymes accountable for glucose metabolism. Persistent hyperglycemia is the major factor responsible for such metabolic alterations that lead to the development of diabetic complications such as neuropathy and micro-vascular complications [
57]. Hepatic Hexokinase and glucose-6-phosphate dehydrogenase activities have been found to be decreased in the diabetic rats, which may be due to the insufficiency of insulin. Hexokinase is one of the important enzyme responsible for phosphorylation of glucose into glucose-6-phosphate [
58].
Insufficiency of hexokinase results in decreased glycolysis and a marked reduction in the utilization of glucose for the production of energy. Oral administration of ALEx to diabetic rats resulted in considerable increase in the activity of hexokinase in dose dependent manner (Table
5).
The activities of hepatic glucose-6-phosphatase as well as fructose-1,6 biphosphatase were increased to a significant extent in STZ induced diabetic rats. The above mentioned enzymes are the key regulators in gluconeogenic pathway. The increased activities of the two enzymes may be due to the increased synthesis of enzymes contributing to the enhanced glucose production by the liver in the period of diabetes [
59]. In our research exertion, administration of ALEx had a significant effect on the level of glucose-6-phosphatase and fructose-1,6 biphosphatase, which decreased to considerable level in dose dependent manner. Maximum effect was observed in 400 mg/kg body weight. The reduction in the above two biochemical enzymes portrays the sequential metabolic correlation between increased glycolysis and decreased glyconeogenesis.
In the pathogenesis of diabetes, lipid plays a significant factor. Increased level of cholesterol and lipids in plasma represent a risk factor for coronary artery disease [
60].
Increased level of total cholesterol, triglycerides, low density lipoprotein cholesterol (LDL-cholesterol), very low density lipoprotein cholesterol (VLDL-cholesterol) was observed in streptozotocin induced diabetic rats. Hypercholesterolemia in the rats received streptozotocin is caused by increased intestinal absorption and increased cholesterol biosynthesis [
54]. Treatment with ALEx reduced the total cholesterol, LDL-c, VLDL-c and triglycerides level to a significant extent in dose dependent manner, while increasing the beneficial HDL level to a considerable extent. It is assumed that ALEx may exert its hypocholesterolemic effect either due to decreased intestinal absorption or decreased cholesterol biosynthesis. The lipoproteins in the diabetic rats are oxidized and may be cytotoxic, which can be reversed by the administration of antioxidants [
61]. Our results clearly demonstrated that ALEx recovered the imbalanced lipid profile of STZ induced diabetic rats in dose dependent manner.
Antioxidant capacity is reduced to a significant extent in the plasma of STZ-induced diabetic rats, due to the higher requirement of antioxidants in order to regulate the reactive oxygen species (ROS) homeostasis [
62]
. Nevertheless, enhanced plasma antioxidant capacity in conjunction with reduced lipid peroxidation could be attained by regular ingestion of rich source of antioxidant compounds. In our research exertion, we examined the antioxidant capacity of ALEx. ROS can be primarily eliminated by essential free radical scavenger enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and glutathione Peroxidase (GSH-Px). As it is obvious from the Table
7 that activities of antioxidant related enzymes were detiorated by administration of streptozotocin (STZ). When the activities of these important antioxidant enzymes were diminished, the superoxide anion and hydrogen peroxide (H
2O
2) radical are available in excess, prompting the production of ROS and dissemination of lipid peroxidation. The level of SOD, CAT, GSH, GSH-Px were diminished in all the tissue of diabetic individuals [
63]. Supplementation of ALEx in STZ induced diabetic rats protect, to certain degree, further improvement in the activities of GSH, GSH-Px, CAT and SOD in liver of the diabetic rats.
Diabetic nephropathy (DN) is one of the major micro-vascular complications of diabetes mellitus. In our present research study, the development of DN is confirmed by significant enhancement in the level of blood urea nitrogen (BUN), glycated serum protein (GSP) and serum creatinine (Scr). Supplementation of ALEx in dose dependent manner improves the renal function parameters. Effect of ALEx 400 mg/kg body weight on reducing oxidative stress and renal function parameters was significantly (p < 0.05) better than the other doses.
Histopathological examination of diabetic pancreas, showed islet of langerhans with fatty infiltration and damaged acini. Administration of ALEx restores the morphological changes in the pancreas to normal. Similarly, the microscopic sections of STZ-diabetic liver demonstrated the damaged central vein and surrounding portal triad. Supplementation of ALEx at different dose recovers the normal histology of liver. Furthermore, the damaged glomeruli, tubules, collecting ducts and ascending and descending limbs were seen the kidney of STZ-induced diabetic rats. These destructive morphological changes were upturned to normal in all ALEx treated groups. Correspondingly, arranged cardiac myocytes were observed in the ALEx supplemented groups as compared to the toxic diabetic rats. According to the microscopic examinations, the severe hepatic, renal, pancreatic and cardiac lesions induced by STZ were significantly diminished and restored by administration of ALEx at lower to higher doses.
Conclusion
The results of the present investigation indicate that ALEx ameliorates the hypoglycemia mediated oxidative stress as well as corrects the lipid profile, hepatic and renal parameters, which was evidenced by improved glycemic control, lipid, renal, hepatic as well as antioxidant biochemical parameters. It can also be concluded that ALEx is a good source of natural antioxidants, which could be a valuable tool in controlling lipid peroxidation and maintaining lipid and lipoproteins. The histological and ultra-structural observations made on the pancreas, liver, kidney and heart tissue substantiate that ALEx protects the oxidative damage of islets of langerhans, hepatocytes, glomeruli and cardiac myocytes on account of its antioxidant potential. Consequently, further studies on the isolation of active principle (s) which exerts the anti-diabetic, hepatic and renal protective effect from ALEx are at the developmental stage in our laboratory.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DA and MS carried out the experimental work, biochemical and statistical analysis. VK, AV & PSG designed and planned the study as well as drafting and revision of the manuscript. HK, VD & VM performed the histological study, interpretation and analysis work. All authors read and approved the manuscript.
This article has been retracted. Please see the retraction notice for more detail: https://doi.org/10.1186/s12906-023-04092-8"